Rapid mass spectrometry methods for antimicrobial susceptibility testing using top-down mass spectrometry

Abstract

Methods and systems for rapid prediction and/or confirmation of antimicrobial susceptibility of a microorganism using top-down mass spectrometry, ion-ion chemistry, and a database with susceptibility, pathogenicity and antimicrobial resistance markers for sample characterization.

1 . A method for predicting antimicrobial susceptibility of a microbe in a sample, the method comprising:
 a) disrupting one or more microbes present in a sample to form a fluid microbial extract;   b) separating a soluble protein fraction from an insoluble protein fraction present in the fluid microbial extract to form a liquid solution of the soluble protein fraction;   c) ionizing a stream or flow of the solution of soluble protein fraction to form one or more ionized proteins, wherein the one or more ionized proteins comprises positively charged ions, the positively charged ions comprising a plurality of ion species;   d) analyzing the plurality of ion species with a mass analyzer of a mass spectrometer system to obtain mass spectral data, searching a database containing mass spectral data of known microbial proteins, and identifying at least one microbe from the database based on the determined mass spectral data of the ion species derived from ionization of the proteins from the sample;   e) from a database containing molecular weights and/or sequence data of the analyte compounds associated with antimicrobial susceptibility, selecting a list of analyte compounds whose presence in the identified microbe is predictive of the state of antimicrobial susceptibility for the identified microbe to form a selected analyte database, the list of analyte compounds comprising protein and/or polypeptide compounds;   f) isolating at least a first subset of the plurality of ion species with the mass spectrometer system, each isolated subset of the at least first isolated subset comprising a respective single mass-to-charge (m/z) ratio or range of m/z ratios, wherein the at least first isolated subset is selected based on the microbe identification and the selected analyte database;   g) generating a plurality of first-generation product ions species from each isolated subset of ion species by causing each said isolated subset of ion species to be reacted with reagent anions that, upon reaction, extract protons from each of one or more ion species of said isolated subset of ion species that comprises a protonated molecule of a protein or polypeptide compound whose charge is reduced by the reaction;   h) acquiring at least one mass spectrum using the mass spectrometer system either of some or all of the first-generation product ion species or of a plurality of second-generation product ion species generated by further reaction or fragmentation of the first-generation product ion species;   i) using m/z ratios of the first-generation or second-generation product ion species to search the selected analyte database; and   j) identifying the presence or absence of at least one analyte compound from the sample predictive of the state of antimicrobial susceptibility for the identified microbe.   
     
     
         2 . The method of  claim 1 , wherein the analyzing in (d) comprises:
 i) acquiring one or more mass spectra representative of the plurality of ion species;   ii) determining molecular weights for the proteins from the one or more mass spectra; and   iii) using the determined molecular weights to search a database containing molecular weights of known microbial proteins and identifying at least one microbe from the database based on the basis of the determined molecular weights of the proteins from the sample.   
     
     
         3 . The method of  claim 1 , wherein the analyzing in (d) comprises:
 i) in a first mass spectrometry step, acquiring one or more first mass spectra representative of one or more of the plurality of ion species;   ii) determining molecular weights for the proteins from the one or more first mass spectra;   iii) using the determined molecular weights to search a database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the database;   iv) in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the of the plurality of ion species and fragmenting the precursor ions by fragmentation means to produce a plurality of MS 2  product ions;   v) using m/z ratios of the one or more precursor ions and/or the plurality of MS 2  product ions to search a database containing molecular weights of known microbial proteins and product ion m/z values of the known microbial proteins; and identifying at least one microbe from the database based on the basis of the determined molecular weights and product ion m/z values of the ionized proteins from the sample.   
     
     
         4 . The method of  claim 1 , wherein the analyzing in (d) comprises:
 i) acquiring one or more mass spectra representative of one or more of the plurality of ion species; and   ii) using the acquired mass spectra and a mass spectral deconvolution program that differentiates signals of the mass spectra to search a database containing mass spectra of known microbial proteins and identifying at least one microbe from the database based on the basis of the acquired mass spectra of the ionized proteins from the sample.   
     
     
         5 . The method of  claim 1 , wherein the generating of the plurality of first-generation product ions species from each isolated subset of ion species in (g) is followed by an ion fragmentation reaction of the PTR product species to obtain a plurality of fragment ion species. 
     
     
         6 . The method of  claim 1 , wherein the microbe is identified as a particular sub-species of a microorganism and the list of analyte compounds includes those that are predictive of the state of antimicrobial susceptibility for the particular subspecies. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein step b) is performed using a solid phase extraction device. 
     
     
         9 . The method of  claim 1 , wherein (a)-(j) are performed using automated instrumentation. 
     
     
         10 . A method for predicting antimicrobial susceptibility of a microbe in a sample, the method comprising:
 a) exposing a sample containing one or more microbes to culture conditions which can induce production of antimicrobial resistance markers to form an induced sample;   b) disrupting one or more microbes present the induced sample to form a fluid extract;   c) separating a soluble protein fraction from an insoluble protein fraction present in the fluid extract to form a liquid solution of the soluble protein fraction;   d) ionizing a stream or flow of the solution of soluble protein fraction to form one or more ionized proteins, wherein the one or more ionized proteins comprises positively charged ions, the positively charged ion comprising a plurality of ion species;   e) analyzing the plurality of ion species with a mass analyzer of a mass spectrometer system to obtain mass spectral data, searching a database containing mass spectral data of known microbial proteins, and identifying at least one microbe from the database based on the determined mass spectral data of the ion species derived from ionization of the proteins from the induced sample;   f) from a database containing molecular weights of antimicrobial resistance markers expressed by microbes, selecting a list of antimicrobial resistance markers associated with the identified microbe species to form a selected marker database, the list of antimicrobial resistance markers comprising protein and/or polypeptide compounds;   g) isolating at least a first subset of the plurality of ion species with the mass spectrometer system, each isolated subset of the at least first isolated subset comprising a respective single mass-to-charge (m/z) ratio or range or m/z ratios, wherein the at least first isolated subset is selected based on the microbe identification and the selected marker database;   h) wherein the at least first isolated subset of ions are reduced in charge state by: i) at least one proton transfer reaction (PTR) to obtain a plurality of PTR product ion species, or ii) at least one PTR to obtain a plurality of PTR product ion species followed by an ion fragmentation reaction of the PTR product ion species to obtain a plurality of fragment ion species;   i) acquiring at least one mass spectrum using the mass spectrometer system of at least some of the PTR product ion species or at least some of the fragment ion species;   j) using m/z ratios of the PTR product ion species or the fragment ion species to search the selected marker database; and   k) determining the presence or absence of at least one antimicrobial resistance marker from the induced sample, wherein the presence or absence of the identified antimicrobial resistance marker in the induced sample as compared to the microbial sample not induced is predictive of antimicrobial susceptibility for the identified microbe.   
     
     
         11 . The method of  claim 10 , wherein the exposing to culture conditions which can induce production of antimicrobial resistance markers is for about 1 minute to about 30 minutes. 
     
     
         12 . The method of  claim 10 , wherein the exposing comprises contacting the sample with at least one antimicrobial agent with a concentration below the resistance breakpoint for the at least one antimicrobial agent. 
     
     
         13 . The method of  claim 10 , wherein the analyzing in (e) comprises:
 i) acquiring one or more mass spectra representative of the plurality of ion species;   ii) determining molecular weights for the proteins from the one or more mass spectra; and   iii) using the determined molecular weights to search a database containing molecular weights of known microbial proteins and identifying at least one microbe from the database based on the basis of the determined molecular weights of the proteins from the induced sample.   
     
     
         14 . The method of  claim 10 , wherein the analyzing in (e) comprises:
 i) in a first mass spectrometry step, acquiring one or more first mass spectra representative of one or more of the plurality of ion species;   ii) determining molecular weights for the proteins from the one or more first mass spectra;   iii) using the determined molecular weights to search a database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the database;   iv) in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the of the plurality of ion species and fragmenting the precursor ions by fragmentation means to produce a plurality of MS2 product ions;   v) using m/z ratios of the one or more precursor ions and/or the plurality of MS2 product ions to search a database containing molecular weights of known microbial proteins and product ion m/z values of the known microbial proteins; and identifying at least one microbe from the database based on the basis of the determined molecular weights and product ion m/z values of the ionized proteins from the induced sample.   
     
     
         15 . The method of  claim 10 , wherein the analyzing in (e) comprises:
 i) acquiring one or more mass spectra representative of one or more of the plurality of ion species; and   ii) using the acquired mass spectra and a mass spectral deconvolution program that differentiates signals of the mass spectra to search a database containing mass spectra of known microbial proteins and identifying at least one microbe from the database based on the basis of the acquired mass spectra of the ionized proteins from the induced sample.   
     
     
         16 . The method of  claim 10 , wherein the microbe is identified as a particular sub-species of a microorganism and the list of antimicrobial resistance markers includes those that are predictive of the state of antimicrobial susceptibility for the particular sub-species. 
     
     
         17 - 19 . (canceled) 
     
     
         20 . A method for confirming a predicted antimicrobial susceptibility of an identified microbe in a sample, the method comprising:
 a) exposing a sample containing an identified microbe with a predicted antimicrobial susceptibility to culture conditions which can induce production of antimicrobial resistance markers to form an induced sample;   b) extracting, from the induced sample, a liquid solution comprising a mixture of sample-derived proteins and polypeptides;   c) introducing at least a first portion of the liquid solution into an ionization source of a mass spectrometer;   d) generating positively charged ions of the mixture of sample-derived proteins and polypeptides, the positively charged ions comprising a plurality of ion species;   e) isolating at least a first subset of the plurality of ion species, each isolated subset of the at least a first isolated subset comprising a respective single mass-to-charge (m/z) ratio or range of m/z ratios;   f) generating a plurality of first-generation product ions species from each isolated subset of ion species by causing each said isolated subset of ion species to be reacted with reagent anions that, upon reaction, extract protons from each of one or more ion species of said isolated subset of ion species that comprises a protonated molecule of a protein or polypeptide compound whose charge is reduced by the reaction;   g) acquiring at least one mass spectrum using a mass analyzer of the mass spectrometer, either of some or all of the first generation product ion species or of a plurality of second-generation product ion species generated by further reaction or fragmentation of the first-generation product ion species;   h) conducting a search of the at least one mass spectrum of either first-generation or second-generation product ion species for a set of one or more m/z ratios that are diagnostic of at least one marker for the predicted antimicrobial susceptibility of the identified microbe;   i) determining the presence or absence of at least one antimicrobial susceptibility marker from the induced sample; wherein presence of an antimicrobial susceptibility marker in the induced sample as compared to the microbial sample not induced confirms the predicted antimicrobial susceptibility of the identified microbe.   
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 20 , wherein the exposing comprises contacting the sample with at least one antimicrobial agent with a concentration below the resistance breakpoint for the at least one antimicrobial agent. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 20 , wherein a rapid phenotypic antimicrobial susceptibility test was used to predict the antimicrobial susceptibility of the identified microbe prior to (a). 
     
     
         25 . The method of  claim 20 , further comprising separating a soluble protein fraction from an insoluble protein fraction present in the liquid solution of (b) prior to the introducing of step (c). 
     
     
         26 . The method of  claim 25 , wherein the separating of the soluble protein fraction is performed using a solid phase extraction device. 
     
     
         27 - 31 . (canceled)